The invention relates to a method for moving a sample substance by a sample feeder from a sample pickup site to a sample delivery site of a sample processor, preferably a sample analyzer.
In a known procedure (German patent document A1 38 05 808, FIGS. 10, 11), the sample substance is withdrawn by a pipetting system from a sample-vial rack and moved to the sample processor in the form of a vertically oriented electrophoresis apparatus. Moreover several samples may be picked up simultaneously using a corresponding number of pipets and be fed into corresponding sample wells at the upper gel edge. This known sample feeder system is mechanically highly complex, the more so when very thin gel layers are being used and at the same time a large number of samples must be analyzed. The gap between the glass plates subtended by the gel thickness presently is less than a mm for the desired gel thicknesses; the separation between consecutive sample sockets is a few mm. Depending on gel width, up to 100 sample wells may be arrayed adjacently. The complexity is correspondingly high for such a number of pipets, and so are the requirements of positional accuracy. In practice the gel can only be oriented vertically because otherwise the sample wells might leak.
A procedure of the initially cited kind is known from the patent document WO 94/11529. The sample substance is chemically specifically bonded to the teeth of the comb-shaped sample feeder. After the sample feeder has been moved to the electrophoresis gel, the specific chemical bond is dissolved by adding an appropriate means (formamide), whereby the sample substance then is able to detach from the sample feeder and able to penetrate the gel under the action of the electric field. The comb-shaped feeder only comprises 8 teeth and accordingly only eight samples can be analyzed simultaneously. This feature may be due to the fact that only comparatively wide teeth allow providing sufficient sample substance.
The objective of the invention is to create a method of the cited kind to allow effective and simple sample transport.
This problem is solved in that the sample feeder comprises at least one porous material element of such pore size that the sample substance is kept in the liquid state by capillary forces in the said porous material at least when the sample is picked up by the sample feeder and when the sample is delivered to the sample processor. Accordingly the sample substance is kept in the liquid state merely by capillary forces during sample pickup or sample delivery. In the interim, the sample substance is present, though not mandatorily, in dried form in the pores of the porous material. In any event complex mechanisms such as multi-pipeting systems are eliminated. As regards sample delivery, the porous material need only be brought into contact with the sample substance in the liquid state; no reactions to chemically bond the sample substance to the sample feeder are at all required, nor are the chemical reactions during sample delivery to dissolve the chemical bond between sample substance and sample feeder. Because the capillary forces keep the sample substance in the liquid state during sample transport until transfer into the sample processor, and this regardless of the direction of gravity, restrictions no longer are incurred on the orientation of the sample processor. The porous material may be laminar and thus may be inserted in problem-free manner between the glass plates of a gel electrophoresis apparatus. At least the material part of the sample feeder consists preferably entirely of a porous material, as a result of which the capacity of the material element for the sample substance shall be relatively high. Therefore it is enough to use small-format material elements to allow a corresponding plurality of compactly configured material elements. For a given width of the electrophoretic gel, the number of the samples which must be analyzed simultaneously can therefore be significantly raised, for instance to 192, even 384 samples.
In a further development of the invention, following sample pickup by the feeder, the sample is dried and the feeder is moved into a liquid phase before the sample is delivered. The sample feeder dried in the interim is especially easily manipulated; the danger of interim contamination is substantially reduced.
To constrain the sample feeder to deliver the sample to the sample processor, the sample feeder can be processed, for instance mechanically, illustratively being mechanically compressed, or compressed air being blown through it. However, in especially preferred manner, an electrical field is generated in the zone of the sample delivery site to cross the material element in order to generate a current of electrically charged molecules, macromolecules or particles of the sample substance from the porous material element into the sample processor. The electric field intensity is selected in such manner as a function of electric charge that the capillary forces are overcome. The use of this method stage is especially advantageous in electrophoresis because it including anyway means for electric field generation.
The sample feeder loaded with the sample substance being especially easily manipulated as indicated above, there follows the advantageous option to move the sample feeder, once loaded with sample, from the sample pickup site to the sample delivery site. Thus the sample can be picked up at a site arbitrarily away from the sample processor apparatus.
Alternatively however the sample also may be moved from the sample pickup site to the sample delivery site in that, provided there be physical connection implemented by the porous material element of the sample pickup site between the sample pickup and delivery sites, the capillary forces shall ensure sample transport. This kind of sample feeding process is used foremost when the sample delivery site is unduly inaccessible or highly compact, for instance as regards microchip sensors, in particular DNA sensors. In such cases a separate material element, independent of the others"" material elements, is used for each sample, so as to avoid cross-talk, that is mixing of sample substance, from the beginning, an event which otherwise, in the light of the comparatively large quantities of sample substance involved, might occur.
However the feasibility of mixing also may be exploited in controlled manner in that the porous material will be wetted at different sites or consecutively at the same site with the substances to be mixed. Illustratively, by appropriate additions, proteins (for instance antigens) may be mixed in the porous material with antibodies, or DNA with complementary hybridization DNA or DNA with labeling means.
Comparatively small amounts of sample substance may be used in the first above alternative of physical transport of the sample feeder from the sample pickup site to the sample delivery site, and consequently the danger of crosstalk in general can be disregarded also where connected material elements are involved.
Because of the above easy handling and the comparatively high capacity for sample substance of the porous material element, a preferred feasibility is offered, namely a sample processor comprising a plurality of sample delivery sites operates with a corresponding plurality of porous material elements.
As already mentioned, separate material elements may be used each time, the method comprising rigorous separation without danger of crosstalk. However the manufacture and handling of the material elements will be substantially simplified if the sample feeder of the invention comprises one material element support bearing the material elements in an array corresponding to the configuration of the sample pickup sites. The sample feeder in this design may be substantially like a comb when the sample pickup sites are confined in linear or arcuate manner.
The sample feeder can be manufactured in especially simple manner, for instance by stamping, if it includes a sheet of porous material comprising all porous material elements.
However the material element support also may bear a corresponding plurality of mutually separate material elements. Such a geometry is preferred when danger of crosstalk cannot be excluded otherwise.
The material element support may be in the form of a substrate sheet or also be of another design, for instance like a clamp.
A porous material with a mean pore size less than 100xcexc, preferably less than 10xcexc, most preferred less than 1xcexc, is used, resp. with a mean pore size between 1.5 and 0.2xcexc, best of all of about 0.5xcexc. Such a porous material offers adequate capillary force for the conventional sample substances used in particular in biochemistry.
It has been found that in particular a porous material made of porous cellulose acetate or porous polyethylene or porous glass or agarose gel or other wide-mesh gel matrices is well suited.
Sample delivery to the sample processor in the presence of said electric field requires that the moved substances be electrically charged. This charge may be generated in biological macromolecules in the required manner by adjusting the pH value of the liquid phase in appropriate manner.
It was discovered that under the effect of the electric field, almost all the sample substance is delivered from the sample feeder to the sample processor. As a result the sample feeder may be used repeatedly in sequence to deliver sample liquid. Again the complexity of implementing the method of the invention is reduced by this feature.
To facilitate loading the sample feeder with a plurality of samples, the invention proposes using a sample-vessel rack comprising a number of sample wells in a geometry corresponding to the configuration of the material elements of the feeder. As a result it is enough that the illustratively comb-shaped sample feeder dips simultaneously with all its comb-teeth into the plurality of sample wells.
The above described method is applicable in a number of sample processors, in particular to detect and/or to manufacture biochemical reaction products. Another application is a mass spectrometer. A preferred application is delivering sample liquids to an electrophoretic or chromatographic apparatus.
The separator, preferably the separation gel of the electrophoretic apparatus, furthermore may be oriented, not only vertically in the heretofore conventional manner, but also horizontally or in any direction.
The invention furthermore proposes that a liquid phase, preferably a buffer solution, be added at one zone at a loading end of a separator of the electrophoretic or chromatographic apparatus, namely before or after the sample feeder has been moved near. In the case of a sample feeder that in the meantime was dried, the liquid phase dissolves the sample substance that was reversibly adsorbed in the pores inside these pores. The resulting capillary forces then retain the sample substance in the pores as long as no external forces, such as the electric field forces of the electrophoretic apparatus, arise. By adding the liquid phase, in particular the buffer solution, the earliest onset of migration of the sample substance can be determined; this will also be the case if the sample substance was not dried in the meantime.
The electric field effect allows immediate transfer of the sample substance into the separator; however, because of the presence of the liquid phase, preferably a buffer solution, a gap up to several mm also can exist between the sample feeder and the loading end of the separator.
The invention further proposes that with respect to a separator appropriate to simultaneously separate several sample liquids and with several sample-liquid pickup sites along an edge of the separator, the sample-liquid pickup sites form sample-liquid wells in the separator to receive corresponding porous material elements of the sample feeder. The sample-liquid wells may be made in this respect independently of the sample feeder and before the sample feeder is brought near, or, alternatively, by seating the comb-shaped sample feeder already fitted with sample substance in the separation gel before latter is polymerized.
The sample-liquid clearances inherently prevent mixing different samples at the edge of the separator.
It was found however that using a separator appropriate for a simultaneous separation procedure and with several sample pickup sites along the edge of the separator, the sample feeder can be made to contact the edge of the separator or be mounted at a gap of several mm from this edge. In this case there are no sample-liquid wells and the separator terminates continuously along the straight or arcuate edge of the separator. This design facilitates the preparation of the separator. It was further discovered that the linear sample density (parallel to the edge of the separator) can be further increased on account of the elimination of the strips of gel between the wells, without thereby degrading measurement, in particular without danger of sample-mixing. Moreover precise, mutually parallel electrophoretic tracks are generated allowing accurate comparative analysis of adjacent tracks, the more so in the invention the beginning of electrophoresis of each track can be triggered with accurate simultaneity. The cause of such significant advantages may be that in the invention, the discharge of sample substance from the sample feeder and hence the entry into the electrophoretic gel is triggered solely by the electric field with constrained migration of the charged particles in the direction of the field. A motion of a different kind, in particular transverse diffusion, is suppressed from the start.
The above described advantages of the invention also apply at least in part when relating to delivering sample liquid to an electrophoretic or chromatographic apparatus comprising one or more separation capillaries. In this case, too, high sample density and simple handling can be achieved.
As already indicated, the method of the invention can be used to deliver sample liquid to a microchip system, in which case the sample feeder(s) also may be stationary separate material elements in order to permanently connect inaccessible sensor zones, for instance 2 mmxc3x972 mm in size, to more accessible delivery sites.
Moreover the invention concerns a sample feeder with which to carry out the above cited method comprising at least one material element of appropriately porous nature.
Furthermore the invention relates to an electrophoretic apparatus comprising a sample feeder to carry out the said method, where, as already discussed, a horizontal configuration, or an arbitrary configuration of the separation gel is possible in addition to the heretofore conventional one.
Another aspect of the invention concerns a method whereby to carry out a reaction between at least a first co-reactant and at least a second co-reactant, preferably to detect and/or to prepare a biochemical reaction product in a sample liquid, said method being characterized in that at least one first co-reactant reversibly adsorbed on a porous material is made to contact at least one second co-reactant in a liquid phase, the pore size of the porous material being such that the liquid phase is retained at least in part in the porous material by capillary forces.
Preferably the reaction in the just-above described method is biochemical, that is it is a reaction in which participate biological macromolecules for instance proteins, glycoproteins and/or nucleic acids or complexes of such macromolecules, for instance immunity complexes between antibodies and antigens, protein/nucleic-acid complexes or nucleic-acid/hybridization complexes as co-reactants or in which such are reaction products. This method is especially well suited for reaction at nucleic acids, for instance an in-vitro transcription, a nucleic-acid sequencing or a nucleic-acid amplification.
In the method described just above, a first co-reactant is used which is reversibly adsorbed on a porous material of appropriate pore size, that is, adsorption on a porous material does not take place by chemical, covalent bonds or by high-affinity interactions (for instance biotin-streptavidin), and accordingly elution of the first co-reactant and/or of the reaction products from the porous material essentially succeeds completely under appropriate conditions.
Moreover several first co-reactants may be adsorbed to the porous material: for instance the material may be impregnated with a liquid containing several co-reactants or the material may be impregnated consecutively or at different sites with several liquids.
In the method of the invention, the porous material may be used in such manner that the first co-reactant is adsorbed to it in dry or moist form. To improve the stability of the adsorbed co-reactant or to improve the volume of reaction, dry adsorption is preferred. Drying of the porous material may be carried out conventionally, for instance by freeze- or vacuum-drying.
In one implementation of the method of the invention, the reaction is a testing reaction to detect an analyte in a biological sample, for instance a sample including a body fluid such as serum, blood, plasma, urine, saliva etc. Other biological samples such as tissue samples also may be used.
In the testing reaction detecting an analyte, the first co-reactant reversibly adsorbed on the porous material preferably is a substance able to specifically bond to the analyte to be determined or it may be a substance competing with the analyte for a further bonding partner. If for instance the analyte is an immunochemically detectable antigen, the first co-reactant may be an antibody bondable to the analyte or an analyte-analogue able to compete with the analyte to bond to an antibody. If illustratively the analyte is a nucleic acid, the first co-reactant may be a nucleic acid complementary to the analyte or a corresponding nucleic-acid/analogue (for instance a peptide nucleic acid).
In detection procedures, a labeling group appropriately is present during the reaction to indicate the occurrence and/or the intensity of the reaction and thereby to allow qualitative or quantitative determination of the analyte. Preferably the labeling group shall be non-radioactive, for instance being a fluorescent or luminescent group, an enzyme, a metal particle, an NMR-active group or another in the field of biochemical detection procedures familiar to the expert.
In a preferred implementation of the invention, the reaction is carried out in such manner that the reaction products are essentially wholly held inside the pores. Also in preferred manner, the reaction products are essentially wholly removable from the porous material. In this manner it is possible to carry out quantitative reaction analysis.
As described above, the sample liquid containing the reaction product may be delivered to a sample processor, preferably a sample analyzer.
Another advantage of carrying out a reaction of the invention in the pores of a porous material is that after the desired time of reaction it is not necessary to add a non-aqueous stop or denaturing reagent such as formamide. As regards the procedures of the state of the art, in particular reactions at nucleic acids, such a formamide reagent must be added to achieve adequate denaturation of the nucleic acids present in the sample. Surprisingly it was discovered that this addition of formamide is unnecessary in the method of the invention.
Another object of the invention is a reagent to carry out a reaction in particular to detect and/or to prepare a biochemical reaction product and comprising at least one part with a material element of porous material, at least one reactive substance being reversibly adsorbed on the porous material and the pore size of this porous material being such that upon contact with liquid, the reactive substance shall be held at least partly in the porous material by capillary forces. This reagent of the invention can be used besides other test components as an ingredient of a reagent kit.
The invention also relates to an electrophoretic apparatus fitted with a porous and preferably comb-shaped sample feeder, comprising a separator, preferably a separation gel, and an electric-field system to apply an electric field to the separator.
Such apparatus is known per se (U.S. Pat. No. 5,464,515 and European patent document A10,493,996). All teeth of the comb-shaped porous sample feeder make bodily contact with the gel at at least one point to assure the transfer of the sample substance (here proteins) into the gel. However heretofore such apparatus did not prove wholly satisfactory.
The objective of the invention is to create an electrophoretic apparatus of the said kind which shall offer improved measurements, in particular resolution.
This problem is solved in that the porous sample feeder used in the electrophoretic apparatus is mounted at a gap larger than zero from the separator and in that the electric-field system generating an electric field is formed within the gap in order to transfer sample substance from the sample feeder into the separator. In the invention therefore the transfer of the sample substance from the porous feeder portion into the separator will not be direct because the sample substance first overcomes the capillary forces by use of the electric field to enter the gap and only then penetrates the separator. The rate of migration of the electrically charged bio-molecule, in particular DNA segments, is substantially larger within the liquid filling the gap than the rate of migration in the separator, in particular separation gel. This causes a stacking effect with spatial concentration of all biomolecules at the edge of the separator in the initial stage of electrophoretic measurement. Extremely sharp bands migrating through the separation gel are present at the sequence end.
However the said gap must always be large enough to exclude direct mechanical contact between the sample feeder and the separator. To preclude crosstalk with neighboring samples, this gap however should not be unduly large. A range of 0.2 to 2 mm was found especially advantageous.
Minimal thicknesses of separator are desired to achieve highest possible sharpness of separation at little substance input. Thus gel thicknesses frequently are 0.5 mm and less. Accordingly extreme care is required to properly insert the occasionally quite flexible, porous, comb-shaped sample feeder between the plates enclosing the separator. The invention suggests in this respect that when using a separator joining two plates, at least one of the plates be beveled on the side of the separator to facilitate inserting the sample feeder. In this way trainees also may be used to insert the sample feeders.
The invention furthermore proposes filling a volume between the sample feeder and the separator with a liquid which is electrically insulating and/or of a higher density than water, preferably with a Ficoll(copyright) or a dextran solution. The accuracy of electrophoretic measurement, in particular the separation sharpness of the bands, is again improved by this step. This feature may be attributed to the increased liquid density impeding migration of the biomolecule out of the porous sample feeder, so that the biomolecules penetrate the liquid only when the electric field is applied, that is at a well defined time. The effect of using the electrically insulating liquid may be that except for biomolecule migration, ion migration does not take place, which otherwise and at least in the vicinity of the bevel might degrade electric field homogeneity and hence also resolution.
The invention also relates to a method for preparing minimal sample volumes from sample materials containing biological macromolecules, preferably such minimal volumes being then picked up by capillary action by a porous and preferably comb-shaped sample feeder.
It is easily seen that the electrophoretic sharpness of separation increases as the sample volume used decreases, namely being concentrated into an ever smaller space for instance at the ends of the comb""s teeth. Only the sensitivity of detection is a lower limit on the measurement technique. However preparing such minute sample volumes for instance of 0.5 xcexcltr is problematical, foremost when using the desired automated pipeting system because such are able to deliver only larger sample volumes in the microliter range. The object of the invention on the other hand is to create a method leading in simple manner to minimal sample volumes, for instance in the range of 0.5 xcexcltr. This method is characterized by the following stages:
(a) preparing an initial sample comprising sample substance and a first volume of a first solvent liquid (preferably water),
(b) preparing an interim sample by adding a second volume, of a second solvent liquid having a lower rate of evaporation than the first solvent liquid, to the initial sample,
(c) evaporating the solvent liquid of the interim sample, the resultant residual volume being the desired sample volume.
Formamide was found to be an especially appropriate solvent, also because it offers further advantageous properties relative to DNA electrophoresis, namely it denatures DNA and can be used as a stop solution. Dextran is also suitable as a second solvent.
In the invention, therefore, a comparatively slight amount of second solvent is addedxe2x80x94the sample substance being soluble in both solvent liquids. The first solvent has been evaporated after stage (c) and only the second solvent containing the sample material remains. The residual volume (which corresponds approximately to the second volume) is therefore independent of the first volume of the first liquid. The residual volume furthermore can be constrained to be less than the second volume if in stage (c) only a portion of the volume of the intermediate sample is used, for instance half. In that case the residual volume will be merely half the second volume. If for instance the initial volume of the initial sample is 4 xcexcltr, and 2 xcexcltr of stop solution consisting of 1 xcexcltr formamide and 1 xcexcltr buffer solution are added, there will be a total of 6 xcexcltr. If half thereof, that is 3 xcexcltr, are delivered into a sample pickup dish and then the whole liquid is evaporated down to the 0.5 xcexcltr formamide portion, there shall be a sample volume of 0.5 xcexcltr.
Evaporation can be accelerated using a blower such as a laboratory fan.
The resulting sample containing formamide can be picked up at once by direct contact with the porous sample feeder because being sucked into it by capillary forces. It was found that the sample feeder so prepared can be sandwiched without requiring further steps before being moved to the electrophoretic apparatus.
Before being inserted into the electrophoretic apparatus, the sample feeder is moistened in another implementation of the invention, preferably using the same liquid that also was used in the gap between the inserted sample feeder and the separator. This pre-moistening of the sample feeder precludes bubble formation in the inserted sample feeder. Such bubbles might interfere with the motion of the biomolecules.
All steps described above per se or in combination provide significantly improved sharpness of separation of individual electrophoretic bands within one electrophoretic track and also allows tracks which are narrower and more tightly adjoining, whereby a larger number of samples can be processed simultaneously in one electrophoretic measurement. Moreover handling is facilitated and it is possible to use dispensing robots.